Multi-drug resistant tuberculosis is increasing in prevalence worldwide; therefore, a greater understanding of the basic biochemistry of Mycobacterium tuberculosis is of utmost importance. Analysis of the M. tuberculosis genome suggests that there may be a biosynthetic pathway analogous to eukaryotic sterol synthesis in this organism. Preliminary evidence indicates that the M. tuberculosis genome encodes enzymes with structural homology to several eukaryotic sterol synthesis enzymes including farnesyl diphosphate synthase, squalene synthase, squalene epoxidase, oxidosqualene cyclase and lanosterol 14a-demethylase. It has been shown that both the M. tuberculosis farnesyl diphosphate synthase and lanosterol 14a-demethylase are functional as well as structural homologs of the eukaryotic enzymes. More importantly, commercial anti-fungal drugs that are known inhibitors of sterol synthesis (specifically oxidosqualene cyclase and lanosterol 14a-demethylase) effectively inhibit the growth of M. tuberculosis in culture. It is hypothesized that M. tuberculosis synthesizes cyclic isoprenoid compounds, perhaps sterols or hopanoids, which are essential to the viability of the organism. Therefore, the specific aims of this proposal are to: 1) identify and characterize cyclic isoprenoid compounds in M. tuberculosis. 2) isolate, enzymatically characterize and determine the essentiality of the sterol synthesis homologs expressed by M. tuberculosis. 3) identify and characterize the active site of the oxidosqualene cyclase homolog. The identification of a sterol/hopanoid biosynthetic pathway in M. tuberculosis and characterization of relevant enzymes represents a novel approach to the identification of previously unsuspected antituberculosis drug targets.